1. Field of the Invention
The present invention relates to a molecular genetic probe and to a method in which such a probe may be used as part of a DNA- or RNA-based assay technique. More specifically, the molecular genetic probe of the invention finds particular use in assay techniques for determining the presence of a virus gene, a gene sequence, or any gene product, e.g., a code for an enzyme, in a suspect sample containing a suspect nucleic acid wherein the suspect nucleic acid is hybridized to the probe and complexed with a labelling agent whose presence can be quantitatively assayed.
2. Description of Background Research and Discussion of Relevant Materials
The present invention provides a technique for assaying for the presence of a viral gene, a gene sequence, or any gene product, e.g., a code for an enzyme. Such techniques are of obvious diagnostic value and are of particular value in kit form such that they can be performed easily and simply by lab personnel having only limited laboratory skills.
There is a particular demand for such techniques, suitable for kits, which are capable of assaying the presence of a particular pathogen; which are specific, and which operate by sensing the presence of nucleic acid specific to the suspect pathogen. Obviously, it is preferable that such techniques be not only accurate, but that they also require performance of a minimum number of manipulations, both by the laboratory technician, as well as in the manufacture of the reagents used in the assay.
It is desirable that the principle of the assay be based upon detection of DNA, or RNA, since such a technique can be used to detect disease, even when symptoms are not apparent. Such techniques are of particular interest in pathogen assays for use in viral diseases wherein one wishes to determine the presence of a particular virus in particular tissue.
In situ techniques exist for assaying for the presence of particular viral DNA and RNA by using hybridization techniques in which probe DNA capable of hybridizing with the suspect viral DNA is radioisotopically tagged and then added to the test specimen. Standard counting and imaging techniques may then be used to quantitatively assay for the presence and amount of suspect DNA which is present.
Such a technique is broadly disclosed in U.S. Pat. No. 4,358,535 to Falkow et al. in which denatured suspect DNA (or RNA), is contacted with single stranded DNA (RNA) probes which have been labelled. A special denaturation technique is disclosed. The probe is selected to hybridize with the denatured suspect DNA. The screening technique is said to be suitable for use in screening viruses (genital Herpes is specifically mentioned), fungi, protozoa, molds, etc. The label used is generally suggested to be a radionuclide, however the patent mentions that in certain instances it may be feasible to employ antibodies which bind specifically to the probe for detection purposes. In such instances the antibodies themselves are labelled. Labels listed include radioactive labels, and ligands which can serve as a specific binding member to a labelled antibody, fluorescers, chemiluminescers, enzymes, antibodies which can serve as a specific binding pair member for a labelled ligand, and the like.
European Patent Application No. 0062286 discloses a method and test kit for the detection of Hepatitis B virus by nucleic acid hybridization. Once again a hybridization probe is used which can then be assayed by a scintillation counting technique.
Russian Patent 649,751 discloses a method for identifying microorganisms by purifying DNA from a microorganism and hybridizing this DNA to DNA from a reference strain. The DNA is labelled with radioactive thymine.
One drawback of such systems is the necessity of handling radioactive materials during the preparation of the probes which is obviously an unsatisfactory situation. Another disadvantage which appears in these techniques is the relatively sophisticated equipment which is necessary for such an enzymatic process.
Both U.S. Pat. Nos. 4,302,204 (Wahl et al.) and 4,139,346 (Rabbani et al.) also disclose hybridization techniques in which RNA and DNA strands are hybridized with labelled probes as a part of a hybridization technique.
It should be noted that DNA per se is not a satisfactory antigen for purposes of the screening technique which is contemplated. The antibody response of viral DNA is often insufficient and not sufficiently specific to render it usable as an antigen.
The modification of DNA and the use of the modified antigen to elicit an antibody response are known techniques.
An article entitled: "Antibodies Specific for Modified Nucleosides: An Immunochemical Approach for the Isolation and Characterization of Nucleic Acids" by Munns and Liszewski, Progress in Nucleic Acid Research and Molecular Biology, Vol. 24, pp. 109-165 (1980), discusses antibodies specific to modified constituents of nucleic acid.
"Immunological Detection of O6-Methylguanine in Alkylated DNA" by Briscoe et al., Biochemistry, Vol. 17, pp. 1896-1901 (1978) describes the potential use of antibodies specific for O6-methylguanine which bind directly to alkylated DNA. A radioimmunoassay system employing these antibodies is said to be considered to be a useful approach in detecting O6-methylguanine in DNA treated with certain alkylating agents.
"Reactivity of Antibodies to DNA Modified by the Carcinogen N-Acetoxy-N-acetyl-2-Aminofluorene", by Sage et al., Biochemistry, Vol. 18, pp. 1328-1332 (1979) describes the reactivity of antibodies to DNA which were modified by N-Acetoxy-N-acetyl-2-Aminofluorene. These antibodies react with the modified DNA, rather than the unmodified form.
"Possible Relevance of O-6 Alkylation of Deoxyguanosine to the Mutagenicity and Carcinogenicity of Nitrosamines and Nitrosamides", by Loveless, Nature, Vol. 223, Jul. 12 (1969) discusses the use of biological alkylating agents such as N-ethyl-N-nitrosourea which modify nucleic acid bases.
Thus, although modification, including ethylation of nucleic acid bases is a recognized phenomenon, the advantages of using this principle in conjunction with hybridization, as part of an efficient assay technique, have not generally previously been appreciated or used to maximum advantage. In fact, while hybridization has been suggested in at least one instance, for use in conjunction with modification in one assay procedure, this procedure does not combine the two techniques to maximum advantage and results in a very cumbersome assay having only limited utility.
Such a technique is seen in European Application S.N. 82301804,9 (published Nov. 13, 1982) which discloses modifying the purine or pyrimidine bases by covalently bonding a moiety consisting of at least three carbon atoms (e.g., biotin) which is capable of forming a detectable complex with a polypeptide when the compound is incorporated into a double stranded RNA, DNA duplex or DNA-RNA hybrid. The polypeptide detectors for the biotin-containing probe may be avidin, streptavidin or an antibiotin immunoglobulin. In particular, the preferred protein for biotin-like probe detection is the antibiotin immunoglobulin. This antibody can be tagged with an enzyme, such as peroxidase. Page 30, line 37 through page 31, lines 1-14 describe the use of an antibody to the biotin constituent. An illustration is provided at page 33 which describes the use of an antibody to detect the biotin or hapten. The IgG-peroxidase example describes an antibody-tagged enzyme detection system. On page 34, lines 25-37 and page 35, lines 1-2, the procedure for utilizing the modified nucleotides to detect the presence of the nucleotide sequences of interest is described. This general protocol can be applied to the detection of nucleic acid sequences of viral, bacterial, fungal or parasitic origins in clinical samples.
As was noted above, this technique is of only limited utility and is extremely cumbersome. Using this technique, nuclease treatment of the template nucleic acid is necessary to nick or open gaps in one of the strands. A biotin substituted base is then inserted into the DNA using polymerase. The modified DNA can only then be complexed with antibiotin which is enzyme tagged to form an antibiotin-enzyme-DNA complex. The double stranded DNA can then be split, with the complexed strand being usable as a probe. Such a technique thus requires that modification occur on DNA in its double stranded form only. The modified single strands can then be used to hybridize only with organisms that contain double stranded nucleic acids, i.e., substances such as single stranded RNA viruses cannot be modified or detected using the approach proposed in this patent application.
Furthermore, the system requires additional reagents such as nuclease and polymerase and the prior synthesis of the biotin base. The use of these substances not only adds to the complexity of the technique but also requires high purity. Also, the use of enzymes which break and then reform the modified nucleic acid chain add to the time and expense involved in manufacturing the kit reagents, again reducing the value of this technique for use in diagnostic kits.
Pages 62-63 of the patent disclose the modification, by addition, of a polynucleotide. Using a mercuric salt, a mercurated derivative is formed. The mercurated derivative is then reacted with a linker arm and a reactive terminal functional group or the modifier itself in the presence of K.sub.2 PdCl.sub.4. Thus, at least two, if not three, steps are required to perform the modification.
Such a procedure is unsatisfactory because it necessarily requires purification between each step. Also, the use of mercury is a strong disadvantage because it renders the process unusable for double stranded systems due to intercalation. Furthermore, as discussed, a linker arm is necessary because of the position on which the moiety is added. A technique which did not require a linker arm would be much more satisfactory. Yet further, using the technique of the publication, only one addition per base is possible, whereas multiple additions per base may sometimes be desirable.